We do know that ASL is a lysis reagent, and that we can increase the ratio of pathogenic:mammalian DNA recovered by performing lysis at a high temperature such as 70 &deg;C. We also know that proteinase K is used to digest proteins, some of which themselves may act as PCR inhibitors or nucleases. After initial digestion and binding, the samples are purified on silica spin columns, which are also well understood.

We do know that ASL is a lysis reagent, and that we can increase the ratio of pathogenic:mammalian DNA recovered by performing lysis at a high temperature such as 70 &deg;C. We also know that proteinase K is used to digest proteins, some of which themselves may act as PCR inhibitors or nucleases. After initial digestion and binding, the samples are purified on silica spin columns, which are also well understood.

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DNA purification columns are composed of porous silica (SiO<sub>2</sub>) beads that have a high surface area:volme ratio. Molecules passing through the column can be selectively retained by both chemical (e.g., charge) and physical (i.e., size) interactions with the porous beads. When nucleic acids are diluted in a high concentration of a chaotropic salt buffer, they will tend to bind to the silica. This is because chaotropic salts (such as guanidine isothiocyanate, present in buffers AL and AW1) disrupt hydrogen-bond organization between water and macromolecules, essentially dehydrating the nucleic acids and causing them to bind to the resin. Ethanol (present in high concentrations in buffers AW1 and AW2) further precipitates the nucleic acids. The column-bound acids are washed with various buffers to remove salts and other contaminants before finally eluting in an ethanol-free, low-salt buffer in which nucleic acids are highly soluble. This final buffer is also at an increased pH to increase charge repulsion between silica and DNA that was previously screened under high salt, low pH conditions. The exact pore size and surface chemistry of the silica beads determine what sizes and kinds of nucleic acid will be bound versus washed away.

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DNA purification columns are composed of porous silica (SiO<sub>2</sub>) beads that have a high surface area:volme ratio. Molecules passing through the column can be selectively retained by both chemical (e.g., charge) and physical (i.e., size) interactions with the porous beads. When nucleic acids are diluted in a high concentration of a chaotropic salt buffer, they will tend to bind to the silica. This is because chaotropic salts (such as guanidine isothiocyanate, present in buffers AL and AW1) disrupt hydrogen-bond organization between water and macromolecules, essentially dehydrating the nucleic acids and causing them to bind to the resin. Ethanol (present in high concentrations in buffers AW1 and AW2) further precipitates the nucleic acids. The column-bound acids are washed with various buffers to remove salts and other contaminants before finally eluting in an ethanol-free, low-salt buffer in which nucleic acids are highly soluble. This final buffer is also at an increased pH to increase charge repulsion between silica and DNA that was previously screened under high salt, low pH conditions. The exact pore size and surface chemistry of the silica beads determine what sizes and kinds of nucleic acid will be bound versus washed away. After purification, two additional steps -- which we'll discuss next time -- can improve downstream performance in PCR.

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After purification, two additional steps can improve downstream performance in PCR. The first is the use of a highly specific polymerase, one with either explicit or inherent hot start properties. Hot start means that it is inactive at low temperature, while the reaction is being set up and initially heated, thus reducing non-specific interactions between primers and non-target DNA. The important point to note here is that the target DNA (DNA of interest) may be present at a low concentration compared to the DNA in the sample as a whole, increasing opportunities for non-specific binding. The second performance enhancer is adding BSA to the PCR. As you saw if you clicked on the Kreader paper, BSA itself binds many inhibitors of PCR, thus acting as a competitor with respect to the polymerase. We would much rather that inhibitors bind the BSA than bind the polymerase and interfere with its function! BSA is hydrophobic and somewhat positively charged, making it a great non-specific binder of proteins that we will use time and again in 20.109.

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We’ve made two minor modifications to Qiagen’s protocol today. One is using a somewhat lower volume of sample than recommended because bird stool is more concentrated than the human stool the kit was designed for. The second is adding chitinase in addition to proteinase K, and incubating at a lower temperature and for a longer time than recommended. Adding chitinase is useful for opening up pathogens with chitin walls, including the fungus microsporidia. You might be wondering why, since you will not use unknown samples for your microsporidia experiment this spring, but rather pre-purified DNA. The reason for this choice is that in practice we have found microsporidia isolation technically challenging, and nor can all bird samples be expected to contain microsporidia. However, your DNA may later be screened by the teaching faculty for analysis in this or a later semester of 20.109. We’re better off having the chance to find microsporidia than not! And empirically, the low temperature/long time incubation increases recovery of bacterial DNA as well.

We’ve made two minor modifications to Qiagen’s protocol today. One is using a somewhat lower volume of sample than recommended because bird stool is more concentrated than the human stool the kit was designed for. The second is adding chitinase in addition to proteinase K, and incubating at a lower temperature and for a longer time than recommended. Adding chitinase is useful for opening up pathogens with chitin walls, including the fungus microsporidia. You might be wondering why, since you will not use unknown samples for your microsporidia experiment this spring, but rather pre-purified DNA. The reason for this choice is that in practice we have found microsporidia isolation technically challenging, and nor can all bird samples be expected to contain microsporidia. However, your DNA may later be screened by the teaching faculty for analysis in this or a later semester of 20.109. We’re better off having the chance to find microsporidia than not! And empirically, the low temperature/long time incubation increases recovery of bacterial DNA as well.

Introduction

Today you will begin your investigation of bacterial composition in different bird populations. Specifically, you will compare gulls from Cordova, Alaska with those that frequent a local reservoir (where exactly in MA?). cont w/ brief background about why this comparison might be of interest and how it fits into more cutting-edge research questions

Your starting point in lab will be an aliquot of bird stool suspended in a viral transport medium (VTM). The VTM helps preserve pathogenic viruses such as influenza for later study. The particular samples that we use today have been screened by the Runstadler lab and confirmed negative for influenza; as interesting as their research in avian flu is, we don't want to become unwitting participants!

To study the bacteria cohabiting with any given bird, we'll attempt to preferentially extract pathogen DNA (as opposed to mammalian DNA) from bird stool samples. We'll then amplify 16S ribosomal RNA gene sequences using the universal primers we discussed last time in a polymerase chain reaction (PCR). The PCR will result in a pool of different 16S sequences representing different species of bacteria present approximately in proportion to their composition in the bird stool. That is, a species that is abundantly present in stool is more likely to have its DNA amplified during the PCR than a rarely present species. The pool of 16S fragments can be cloned into a DNA vector and transformed into laboratory bacteria to produce isolated colonies. Each colony should contain identical copies of 16S DNA (i.e., representing a single species of bacteria), thus allowing us to deconvolve our pool of DNA and analyze individual sequences. Briefly, we'll perform a second DNA extraction from several colonies per original bird stool sample and find out the sequence of each DNA through a method that resembles PCR. The individual steps will make more sense as we complete each of them, and an overview of the process as a whole is shown below.

Make overview schematic for lab progression to accompany above paragraph!

Returning to today's specific work, each of you will extract a DNA pool from a single bird stool sample using a QIAamp stool kit. Stool is a somewhat vexing material from which to extract DNA, because many enzyme inhibitors (including materials that inhibit polymerase) are present. As described in the paper by Carol Kreader, inhibitors in feces include bile salts, and environmental inhibitors (humic compounds present in water and dirt) might also concern us given how the bird stool was collected. Finally, chemicals that degrade DNA may be present, which is especially troubling when one wants to amplify a low concentration DNA. The Qiagen kit contains two reagents that degrade or bind up inhibitors – buffer ASL and the InhibiTEX tablets – but unfortunately their exact contents and mechanisms of action are propietary.

We do know that ASL is a lysis reagent, and that we can increase the ratio of pathogenic:mammalian DNA recovered by performing lysis at a high temperature such as 70 °C. We also know that proteinase K is used to digest proteins, some of which themselves may act as PCR inhibitors or nucleases. After initial digestion and binding, the samples are purified on silica spin columns, which are also well understood.

DNA purification columns are composed of porous silica (SiO2) beads that have a high surface area:volme ratio. Molecules passing through the column can be selectively retained by both chemical (e.g., charge) and physical (i.e., size) interactions with the porous beads. When nucleic acids are diluted in a high concentration of a chaotropic salt buffer, they will tend to bind to the silica. This is because chaotropic salts (such as guanidine isothiocyanate, present in buffers AL and AW1) disrupt hydrogen-bond organization between water and macromolecules, essentially dehydrating the nucleic acids and causing them to bind to the resin. Ethanol (present in high concentrations in buffers AW1 and AW2) further precipitates the nucleic acids. The column-bound acids are washed with various buffers to remove salts and other contaminants before finally eluting in an ethanol-free, low-salt buffer in which nucleic acids are highly soluble. This final buffer is also at an increased pH to increase charge repulsion between silica and DNA that was previously screened under high salt, low pH conditions. The exact pore size and surface chemistry of the silica beads determine what sizes and kinds of nucleic acid will be bound versus washed away. After purification, two additional steps -- which we'll discuss next time -- can improve downstream performance in PCR.

We’ve made two minor modifications to Qiagen’s protocol today. One is using a somewhat lower volume of sample than recommended because bird stool is more concentrated than the human stool the kit was designed for. The second is adding chitinase in addition to proteinase K, and incubating at a lower temperature and for a longer time than recommended. Adding chitinase is useful for opening up pathogens with chitin walls, including the fungus microsporidia. You might be wondering why, since you will not use unknown samples for your microsporidia experiment this spring, but rather pre-purified DNA. The reason for this choice is that in practice we have found microsporidia isolation technically challenging, and nor can all bird samples be expected to contain microsporidia. However, your DNA may later be screened by the teaching faculty for analysis in this or a later semester of 20.109. We’re better off having the chance to find microsporidia than not! And empirically, the low temperature/long time incubation increases recovery of bacterial DNA as well.

Protocols

Part 1: DNA extraction from bird stool, initiate

The following protocol requires many tube changes. Be sure that each tube is clearly labeled with your sample number to avoid swapping samples with your partner. There are a few other steps you can take to avoid cross-contamination: switch pipet tips at every step, keep only one tube open at a time, and avoid getting liquids on the lip of any tube or column.

Before beginning this protocol, check the maximum spin speed of your centrifuge. Some centrifuges reach 20,000 g and others reach only 16,000 g, and the time for centrifugation will have to be adjusted proportionally. Note that rpm stands for rotations per minute while rcf stands for "relative centrifugal force." It is rcf that is equivalent to g-force, not rpm, because different sized rotors will impart different forces at the same rotational speed.

Obtain your 100 μL bird stool sample from the teaching bench ice bucket, according to the number you are assigned on today's Talk page.

Work quickly and keep the sample on your ice bucket until you have finished adding the lysis reagent.

Immediately add 1.4 mL of the lysis reagent (called buffer ASL) and vortex for ~ 1 min, until the solution is homogeneous.

The fastest way to add the appropriate amount of ASL is to add 0.7 mL twice; that way you don't have to rotate the pipet setting in between additions.

A few insoluble particulates may remain. Try vortexing for another 20-30 sec interval up to four more times, and stop vortexing when the sample no longer visibly changes over that interval.

Heat at 70 °C for 5 min on the heat block at the front bench.

Vortex for 15 sec and centrifuge for 1 min at 20,000 rcf or 1.5 min at 16,000 rcf. Place your tubes so that weight is equally distributed in the centrifuge.

Unfortunately, your centrifuges cannot be set for 1.25 min exactly.

Transfer 1.2 mL of supernatant into a fresh 2 mL tube.

Be sure to use the special 2 mL eppendorfs here and not the standard 1.5 mL eppendorf tubes.

Hold the foil-covered InhibitEX tablet over the tube, and gently push until the tablet pierces through the foil and falls into the tube.

Vortex until completely dissolved, which takes about 3 min for these samples.

In the meantime, trim the cap off a fresh 1.5 mL eppendorf tube using small scissors that have been wiped down with 70% ethanol. Prepare a sticky label (in your team color) for the top: write the date and your sample identification number. You should also label the side of each tube, at least with short unique identifier, so you don't lose track of which sample is which in the following step.